THE JOURNAL OF COMPARATIVE NEUROLOGY 278:591-603 (1988) Brainstem Branches From Olivocochlear Axons in Cats and Rodents M.C. BROWN, M.C. LIBERMAN, T.E. BENSON, AND D.K. RYUGO Departments of Physiology and Otolaryngology (M.C.B., M.C.L.), and Department of Anatomy and Cellular Biology (T.E.B., D.K.R.), Harvard Medical School, Boston, Massachusetts 02115; Center for Hearing Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 (D.K.R.); Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114 (M.C.B., M.C.L., T.E.B., D.K.R.) ABSTRACT Horseradish peroxidase was used to label axons of olivocochlear (OC) neurons by intracellular injections in cats and extracellular injections in rodents. These axons arise from cell bodies in the superior olivary complex and project to the cochlea. En route to the cochlea, the thick axons (> 0.7 pm diam.) of medial olivocochlear (MOC) neurons formed collaterals that terminated in the ventral cochlear nucleus, the interstitial nucleus of the vestibular nerve (in cats), and the inferior vestibular nucleus (in rodents). The thin axons (< 0.7 pm diam.), presumed to arise from lateral olivococh- lear (LOC) neurons, did not branch near the CN. Within the CN, the MOC collaterals tended to ramify in and near regions with high densities of granule cells, regions also associated with the terminals of type I1 afferent sons (Brown et al.: J. Comp. NeuroL 278.581-590, '88). These results suggest that those fibers associated peripherally with outer hair cells (MOC efferents and type I1 afferents) are associated centrally with regions contain- ing granule cells, whereas those fibers associated with inner hair cells peripherally (LOC efferents and type I afferents) are not. Key words: cochlear nucleus granule cell, vestibular nucleus, hair cell, axon collateral, efferent nerve Rasmussen ('46, '53) published the initial descriptions of The present study concentrates on the morphology of the the olivocochlear bundle (OCB) from its sources in the su- OC collaterals within the brainstem. These collaterals have perior olivary complex to its presumed endings in the coch- not been described systematically, although they are known lea. More recent work (Warr and Guinan, '79; Guinan et to innervate the ventral cochlear nucleus WCN; Rasmus- al., '83; White and Warr, '83) has divided the olivocochlear sen, '60, '67; Liberman and Brown, '86). We used horserad- (OC) neurons into two systems according to soma location ish peroxidase (HRP) to label collaterals of OC neurons: with respect to the medial superior olive: the medial olivo- intracellular injections for physiologically characterized cochlear (MOC) system and the lateral olivocochlear (LOG) single neurons, and focal extracellular injections for small system. Peripherally, the thick myelinated axons of the populations of neurons. Both types of injections can also MOC neurons terminate mainly on outer hair cells, whereas label primary afferent neurons in the same tissue (Liber- the thin and apparently unmyelinated axons of LOC neu- man and Brown, '86; Brown et al., '88a). Thus, the methods rons terminate predominantly on afferent dendrites just permit us to directly compare the trajectories and termina- beneath inner hair cells (Liberman, '80; Ginzberg and Mo- tions of afferent and efferent fibers in the CN. These de- rest, '83; Guinan et al., '83; Liberman and Brown, '86; scriptions are relevant to an understanding of the Brown, '87). The anatomical differences between the MOC relationship of the feedback system to the afferent and LOC systems suggest at least two general types of pathways. feedback control of the periphery. At present, however, there is no consensus as to the function of either OC system in the awake, behaving animal. Further studies of the anatomical and physiological characteristics of the efferent systems may offer clues to their functional significance. Accepted June 29,1988. 0 1988 ALAN R. LISS, INC. M.C. BROWN ET AL. MATERIALS AND METHODS RESULTS Intracellular injections Classification and general brainstem course of OC Single OC units were recorded in the internal auditory fibers meatus from the region near the vestibulocochlear anasto- In seven cats, nine OC fibers were labeled following intra- mosis in seven Dial-anesthetized (0.73 mag, i.p.) cats as cellular injection near the vestibulocochlear anastomosis. described previously (Liberman and Brown, '86). At the Labeled cell bodies were found in brainstem loci that have time of recording, fibers were classified as "efferent" on the been described as the origins of the medial olivocochlear basis of 1) position within the nerve bundles, 2) regularity (MOC) cell group (Warr and Guinan, '79; Guinan et al., '83). of the interspike intervals, and 3) latency of responses (5- Single OC units respond to monaural sounds presented to 50 msec) to tone bursts (Liberman and Brown, '86). Neurons either the ipsilateral or the contralateral or to both ears were labeled by iontophoresis of HRP, and injections were and have tuning curves with a well-defined characteristic limited to one or two fibers per animal. After a survival frequency (Liberman and Brown, '86). The units labeled in time of about 30 hours, the anesthetized cats were perfused this study ranged in characteristic frequency from 1.9 to 21 intracardially with glutaraldehyde and paraformaldehyde. kHz; six responded only to ipsilateral sound, and three The cochlear nucleus was either separated from the brain- responded only to contralateral sound. stem and processed with diaminobenzidine (Fekete et al., The MOC axons course from their cell bodies (Fig. 1)in+ a '84) or was left attached to the remainder of the brainstem, dorsal direction, approaching the surface of the brainstem sectioned on a freezing microtome, and processed with te- just beneath the fourth ventricle. As the axons travel lat- tramethylbenzidine (Mesulam, '82). Axons in material pro- erally after approaching the midline, they follow the vestib- cessed with tetramethylbenzidine could be traced for long ular nerve root and run ventromedial to the anteroventral distances and their branching patterns could be recon- cochlear nucleus (AVCN). Within the vestibular nerve root, structed, but the crystalline nature of the label prevented MOC axons produce collaterals. The MOC axons cross the detailed analysis of fiber or terminal morphology. For fine inferior vestibular ganglion, enter the auditory nerve (at resolution of the terminal regions, material processed with the vestibulocochlear anastomosis), and terminate periph- diaminobenzidine was used. erally on outer hair cells (Liberman and Brown, '86). The MOC axons are thick (about 2-4 pm diameter in the vestib- Extracellular injections ular nerve root) and have periodic constrictions that we Extracellular injections of HRP were made into the spiral interpret as nodes of Ranvier (Fekete et al., '84; Liberman ganglion region as described previously (Brown et al., '88a). and Oliver, '84). Although thin unmyelinated efferent ax- Data described are from five gerbils (Meriones unguiculu- ons are also present at the recording site in the vestibulo- tus) and eight CD-1 mice (Mus musculus). All injections cochlear anastomosis (Arnesen and Osen, '841, they have labeled both efferent and afferent fibers. Rodent material not been labeled intracellularly, probably because they are was processed with diaminobenzidine. too thin to be impaled with micropipette electrodes. Pre- Electron microscopy sumably, these thin axons are from LOC neurons and ter- minate peripherally near inner hair cells (Brown, '87). Electron microscopic observations were made from brain- In rodents, extracellular injections of HRP made into the stems in two mice. After a brief intracardiac saline rinse, spiral ganglion labeled both thin and thick efferent axons, fixation was achieved by perfusion of a mixture of 0.5% as well as afferent axons. The reaction product within the paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodyl- efferent axons typically faded before reaching the cell body. ate buffer (pH 7.3), followed by 0.5% paraformaldehyde and These partially reconstructed fibers were identified as effer- 3% glutaraldehyde in cacodylate buffer. After 1-hour im- ent because their course was similar to the course of fully mersion in the stronger fixative, the brainstem was dis- reconstructed efferents labeled by intracellular injections. sected and stored overnight in the 0.1 M cacodylate buffer. Medially, the efferent fibers could be retrogradely traced as The tissue was sectioned at 50 pm on a Vibratome. Histo- far centrally as the ventral boundary of the vestibular logical processing for HRP was as described by Fekete et nuclei (Fig. 2, panels 5-71. Laterally, they follow the vestib- al. ('84) with the addition of 1%dimethylsulfoxide to all ular nerve root, which forms the ventromedial boundary of solutions containing cobalt or diaminobenzidine. The sec- the VCN, and exit the brain in the vestibular nerve. Near tions were then stained en bloc with osmium tetroxide and the inferior vestibular ganglion, the efferent fibers cross to uranyl acetate, dehydrated, and embedded in Epon. La- the auditory nerve at the vestibulocochlear anastomosis. In beled fibers and reference marks such as blood vessels were the cochlea, they cross the spiral ganglion and enter the traced from the Epon-embedded tissue sections at the light intraganglionic spiral bundle before disappearing into the microscopic level with the aid of